Antimitotic eleuthesides

ABSTRACT

This invention provides analogs of eleutherobin and the eleuthesides modified at the C-11 position or comprising an epoxide functionality from C-11 to C-12. C-11 to C-12 is an ideal location for conjugating functional moieties to the eleutherobin pharmacophore without significant loss of antimitotic activity. Moieties that may be conjugated at C-11 include those intended to increase the solubility of the pharmacophore, to facilitate drug formulation, or to facilitate in vivo delivery or targeting.

This application is a 371 of PCT/CA02/00944, filed Jun. 25, 2002.Acknowledgement is made of Applicant's claim for priority via U.S.Provisional application 60/299,788, filed on Jun. 22, 2001.

BACKGROUND OF THE INVENTION

Antimitotic compounds interfere with the dynamic assembly anddisassembly of α- and β-tubulin into microtubules causing cells toarrest in mitosis. Prolonged arrest in mitosis eventually leads to celldeath, often by apoptosis. Two chemical classes of antimitotic agents,the vinca alkaloids (vinblastine, vincristine, and vinorelbine) and thetaxanes (paclitaxel and docetaxel), are clinically useful anticancerdrugs. Most known antimitotic agents induce mitotic arrest by inhibitingthe polymerization of tubulin into microtubules. This is the mechanismof the vinca alkaloids and rhizoxin.

Paclitaxel was the first chemical entity shown to cause mitotic arrestby stabilizing microtubules against depolymerization. Four additionalchemotypes that have paclitaxel-like effects were later identified.These include the myxobacterium metabolites epothilones A and B, themarine sponge metabolites discodermolide, laulimalide, andisolaulimalide, and the soft coral terpenoid, eleutherobin (shownbelow). Ojima et al. (1999) Proc. Natl. Acad. Sci. USA 96:4256–4261,proposed a common pharmacophore for the microtubule stabilizingcompounds that effectively accommodates nonataxel, paclitaxel,discodermolide, eleutherobin, and the epothilones. This model predictsthat three regions of eleutherobin (boxes A, B, and C below) areimportant for binding to tubulin (Me=methyl; Ac=acetyl).

Eleutherobin Pharmacophore

The majority of known antimitotic natural products were initiallyisolated because they exhibited potent in vitro cytotoxicity. Onlysubsequent detailed mechanism of action studies revealed that theyarrested cells in mitosis and interfered with tubulin assembly anddisassembly dynamics. For example, rhizoxin is a 16-membered ringmacrolide first isolated in 1984 and determined to be very cytotoxic.Only later was rhizoxin shown to cause the accumulation of cells inmitosis. Sarcodictyins A-D were the first members of a cytotoxicterpenoid class of compounds to be identified (see: D'Ambrosio, M., etal. (1987) Helv. Chim. Acta. 70:2019–2027; and, (1988) Helv. Chim. Acta.71:964–976), their paclitaxel-like properties being recognized onlylater.

Eleutherobin, a diterpene glycoside, was originally isolated from thesoft coral Eleutherobia sp. (possibly E. albiflora) collected in WesternAustralia (see: Lindel, T. et al. (1997) J. Am. Chem. Soc.119:8744–8745; and, international patent application published May 23,1996 under WO 96/14745). Subsequently, eleuthesides A and B wereisolated from a different species of Eleutherobia (E. aurea). Theeleuthesides share the pharmacophore shown above but differ fromeleutherobin by the presence of a hydroxyl substituent at the C-4position of eleutherobin (rather than a methoxyl substituent) and by thepresence of an acetyl group at the 3″ or the 4″ position of thearabinose moiety shown above, in addition to an acetyl at the 2″position (Ketzinel, S., et al. (1996) J. Nat. Prod. 59:873–875). Later,a total synthesis of eleutherobin and eleuthesides A and B was reported(Nicolaou, K. C., et al. (1998) J. Am. Chem. Soc. 120:8674–8680). Asreported in the latter reference, the eleuthesides may be made byconverting C-4 ketal precursors to C-4 hydroxyl forms. Eleutherobin andthe eleuthesides are hydrophobic compounds.

International patent application published May 31, 2001 under WO01/38339 disclosed use of a cell-based antimitotic assay whichdemonstrated potent antimitotic activity in extracts of various marineorganisms. Microscopic examination of cells arrested in mitosis by someextracts showed tubulin bundling, similar to the effects of paclitaxel.Bioassay guided fractionation of these extracts led to the isolation ofeleutherobin and other antimitotic diterpenes, includingdesmethyleleutherobin, desacetyleleutherobin, isoeleutherobin A,Z-eleutherobin, caribaeoside, and caribaeolin.

WO 01/38339 disclosed that certain alterations outside the Ojima, et al.[supra] pharmacophore binding regions resulted in changes in antimitoticpotency. It was also shown that alterations at C-11 to C-13 ofeleutherobin (region B of the pharmacophore) could significantlydecrease potency, as shown by the measured antimitotic activities forcaribaeoside and caribaeolin. Thus the prior art indicates thatmodifications to region B of the pharmacophore for eleutherobin or theeleuthesides should not be made if one wishes to retain antimitoticactivity.

SUMMARY OF THE INVENTION

This invention is based on the discovery that modifications to region Bof the eleutherobin pharmacophore need not be detrimental to antimitoticactivity. As a result of this invention, region B of the pharmacophorebecomes the location of choice for modification of eleutherobin and theeleuthesides or analogs thereof, where such modification is to affectthe characteristics of the antimitotic compound for purposes ofpharmaceutical formulation or to improve delivery of the antimitoticcompound to a target in a patient.

This invention provides novel compounds suitable for use as antimitoticagents and as intermediates for the production of antimitotic agents.The novel compounds of this invention are based on modification inregion B of the pharmacophore which permit retention of antimitoticactivity. Antimitotic compounds of this invention or compounds made fromcompounds of this invention as intermediates can provide improvedtolerance in patients at therapeutically effective doses (improvedtherapeutic index) through better delivery of an effective dose of thecompound to tissues or cells within a patient by the compound acting asa prodrug, or by the compound being joined to another therapeutic agent.

This invention provides an antimitotic compound and pharmaceuticalpreparations thereof, wherein the compound is a compound of formula I orII:

In compounds of this invention, Me is methyl; R¹, R² and R³ areindependently selected from the group consisting of H and a C₁–C₆ acyl;and, R⁴ is selected from the group consisting of H, Me and a substitutedor unsubstituted straight-chain, branched, or cyclic C₁–C₁₀ alkyl.Preferably, an acyl in R¹–R⁴ is acetyl and an alkyl in R¹–R⁴ is a C₂–C₅straight-chain or branched moiety. Any or all of R¹–R⁴ may be selectedindependent of whether the compound is of formula I or II and theidentify of X in formula I.

Preferred embodiments of this invention include those in which: R¹ andR² are H and R³ is Ac; R⁴ is H, ethyl, propyl, butyl or pentyl; one ofR¹ and R² is H and the other Ac; and, R³ is H. In other preferredembodiments: R⁴ is Me; R¹ and R³ are H; and, R² is Ac.

In compounds of formula I of this invention, X is a moiety other thanmethyl, bound through one carbon atom directly to the C-11 position.Thus, X cannot consist of methyl or hydrogen, or comprise a heteroatomjoined directly to C-11. X may be any functional moiety selected toprovide a physical characteristic (such as solubility), to provide achemical characteristic (such as reactivity with another moiety to bejoined to a compound of formula I), or to provide a desired performancein vivo (such as enhanced circulation longevity or targeting to aparticular cell or tissue).

In compounds of formula I, X may be selected from the group consistingof: —CH₂OR; —COR; —COY; —C(OR)R; —CRCR₂; and, —CH₂W. R may be selectedfrom the group consisting of: H; a linear, branched, or cyclic,saturated or unsaturated alkyl group containing one to ten carbons thatmay be substituted with Z; an aromatic group such as phenyl, napthyl,anthracyl, phenanthryl, furan, pyrrole, thiophene, benzofuran,benzothiophene, quinolinc, isoquinoline, imidazole, thiazole, oxazole,or pyridine and the aromatic group may be substituted with Z; and,arylalkyl (ArR*) where the alkyl group (R*) may be linear, branched, orcyclic, and saturated or unsaturated containing one to ten carbons thatmay be substituted with Z and the aryl group (Ar) may be phenyl,napthyl, anthracyl, phenanthryl, furan, pyrrole, thiophene, benzofuran,benzothiophene, quinoline, isoquinoline, imidazole, thiazole, oxazole,or pyridine that may be substituted with Z. Z may be selected from thegroup consisting of: —OH, —OR, —O₂CR, —SH, —SR, —SOCR, —NH₂, —NHR,—NHR₂, —NHCOR, —NRCOR, —I, —Br, —Cl, —F, —CN, —CO₂H, —CO₂R, —CHO, —COR,—CONH₂, —CONHR, —CONR₂, —COSH, —COSR, —NO₂, —SO₃H, —SOR, and —SO₂R (withR being as defined above). Y may be selected from the group consistingof: —OH, —NH₂, —NHR, —NR₂, —SH, —SR and —OR (with R being as definedabove). W may be selected from the group consisting of: R, F, Cl, Br, I,—OSO₃R, —SO₃R, —OPO₃R₂, —PO₃R₂, —SR, —SOR, —SO₂R, —NR₂, —NOR, and —NR₃⁺, with R being as defined above. In R*, and when R comprises an alkylgroup, —CH₂— may be replaced by —O₂S or NR, and CH may be replaced by N.Each R may be the same or different.

Preferred compounds of this invention are compounds of formula I inwhich X is as defined above with R being H, or a linear, branched orcyclic, saturated or unsaturated alkyl group and Y being: —OH, —NH₂,—NHR, —NR₂ or —OR; or, compounds of formula II. In some preferredcompounds of formula I, R is H or an alkyl group, which is preferably aC₁–C₆ branched or straight chain moiety.

A compound of this invention includes salts (preferably pharmaceuticallyacceptable salts) and also includes isomers, including those of the Zand E configurations, those of the α and β configurations at theglycosidic bond and the α and β configurations of the epoxide at C-11 toC-12 in formula II.

This invention also provides the use of a compound or a pharmaceuticalpreparation of this invention as an antimitotic agent and for thepreparation of antimitotic agents including medicaments. This inventionalso provides a method for causing mitotic arrest in one or more cellspresent in a cell population, comprising treating the cell populationwith a sufficient amount of a compound or pharmaceutical preparation ofthis invention to arrest mitosis in one or more cells in the cellpopulation. The cell population may be a population of cancerous cells,including a tumor. This method may be performed in vitro or may beperformed in vivo through administration to a human or animal patientwith a cancer.

DETAILED DESCRIPTION

The structure of compounds of this invention is of a broad scope in asmuch as substituent X in compounds of formula I may be any moietyselected to confer a desired characteristic different from that providedwhen a methyl group is joined at C-11 (as is the case for the naturallyoccurring compounds). Moiety X will be joined to the pharmacophorethrough a carbon atom bound directly to C-11. This means that C-17 foundin the naturally occurring compounds is retained, but is not methyl.Moiety X may be a polar group selected to increase the solubility of thecompound. Such a polar group includes an ionizable group, which wouldfacilitate the formation of salts, such as pharmaceutically acceptableacid-addition salts. Increasing the solubility of antimitotic compoundsof this invention facilitates formulation of the compounds therebypermitting one to avoid difficulties associated with formulation ofhydrophobic drugs. One example of such a substituent joined at C-11 asdescribed in the Examples below is —COH or —COOH. The latter substituentis ideally suited for forming salts.

Moiety X of compounds of formula I of this invention may be selected toincrease the reactivity of region B of the pharmacophore, therebypermitting compounds of this invention to function as intermediates inthe preparation of compounds in which other chemical moieties are joinedto the pharmacophore at region B. Examples of moiety X as described inthe Examples below, which increase the reactivity of region B forjoining to other moieties while retaining stability include: —CNH₂ and—COOH. The latter substituents are ideally suited for joining to amineor carboxylic acid-containing moieties by means of a peptide linkage.Likewise, compounds of formula II of this invention represent stableantimitotic compounds having a modification in region B that facilitatesconjugation to another moiety (see Example 9 below).

Moiety X of compounds of formula I may be a substituent which comprisesa linker which in turn may be used for conjunction to another functionalgroup. Such a linker may be any linker known in the art for joiningbiologically active compounds or for joining a biologically activecompound to a carrier. Such linkers may be cleavable upon the action ofan agent present at or near a target site (e.g. reduced pH) or which isadministered in conjunction with the compound of this invention. Anexample of such linkers are those described by Czerwinski, et al. (1998)Proc. Natl. Acad. Sci. 95:11520–11525, in WO 89/11867 and WO 91/12023,or the metal chelating linkages described in WO 00/64471, WO 01/28569,and U.S. Pat. No. 6,087,452.

Compounds of this invention include compounds intended for use asintermediates (for example to be joined to another chemical moiety) andinclude the resulting products in which moiety X is joined to C-11 as incompounds of formula I. Thus, moiety X of compounds of formula I may beany functional group or moiety selected to provide a desired performancein vivo. Without limitation, moiety X may comprise a peptide (includingpolypeptides and proteins), a lipid, a polysaccharide, apharmaceutically compatible polymer or another drug. Thus, compounds offormula I of this invention include compounds that are conjugated to alipid in a lipid-based delivery vehicle such as a liposome, to a peptidethat facilitates transfer across a cell membrane, to an antibody havingspecificity for a target cell and to a peptide ligand capable of bindingto a cell surface receptor or the like. In other embodiments, moiety Xcomprises a polymer suitable for incorporation within a pharmaceuticalpreparation or which enhances delivery of an active compound within thebody.

Moiety X is joined to a compound of formula I at the C-11 positionthrough at least one carbon atom bound directly to C-11. Such directbinding of the carbon atom to C-11 is by a covalent bond. However, theremainder of moiety X may comprise components which are joined togetheror to the carbon atom that is bound to C-11, through any form ofconjugation. By the terms “conjugation” and “conjugated” it is meantthat components are joined by any of covalent bonds, coordinate covalentbonds, ionic bonds and hydrogen bonds.

The physical, chemical, or biological characteristics of thepharmacophore can be altered in many ways that would be apparent topersons skilled in the art. Different functional groups will alter thesolubility of the pharmacophore through the addition of groups that forexample alter polarity and/or the ability to form hydrogen bonds.Similarly a functional group may alter the stability of thepharmacophore by changing the serum half-life or by controlling therelease of the pharmacophore from a micelle at the target site orconverting a prodrug to the active form at the target site. Further afunctional group may alter the biocompatibility of a pharmacophore forexample by minimizing the side effects of the drug to the patient. Afunctional group may further enhance delivery and targeting of thepharmacophore through the addition of a functional group capable ofbinding the target cells or tissues or facilitating the transport intothe target cells. The functional group may also enhance the anti-tumoractivity of the pharmacophore if for example the pharmacophore isconjugated to another anti-proliferative drug. A person skilled in theart will appreciate what type of functional groups might be added toachieve the desired result in administering the pharmacophore to thepatient and thereby improving the overall therapeutic index.

A functional group conjugated to the pharmacophore may be a biologicaltargeting molecule that binds to a specific biological substance orsite. The biological substance or site is the intended target of thedelivery and targeting molecule that binds to it, enabling the deliveryof the pharmacophore to the tissue or cells of interest.

A ligand may function as a biological targeting molecule by selectivelybinding or having a specific affinity for another substance. A ligand isrecognized and bound by a specific binding body or binding partner, orreceptor. Examples of ligands suitable for targeting are antigens,haptens, biotin, biotin derivatives, lectins, galactosamine andfucosylamine moieties, receptors, substrates, coenzymes and cofactorsamong others. A ligand may include cancer and tumor antigens such asalpha-fetoproteins, prostate specific antigen (PSA) and CEA, cancermarkers and oncoproteins, among others. Other substances that canfunction as ligands for delivery and targeting are certain steroids,prostaglandins, carbohydrates, lipids, certain proteins or proteinfragments (i.e. hormones, toxins), and synthetic or natural polypeptideswith cell affinity. Ligands also include various substances withselective affinity for ligators that are produced through recombinantDNA, genetic and molecular engineering.

Another type of targeting molecule is an antibody, which term is usedherein to include all classes of antibodies, monoclonal antibodies,chimeric antibodies, Fab fractions, fragments and derivatives thereof.Other targeting molecules include enzymes, especially cell surfaceenzymes such as neuraminidases, plasma proteins, avidins, streptavidins,chalones, cavitands, thyroglobulin, intrinsic factor, globulins,chelators, surfactants, organometaffic substances, staphylococcalprotein A, protein G, cytochromes, lectins, certain resins, and organicpolymers. Targeting molecules may include peptides, including proteins,protein fragments or polypeptides which may be produced synthetically orthrough recombinant techniques known in the art. Examples of peptidesinclude membrane transfer proteins which could facilitate the transferof the pharmacophore to a target cell interior or for nucleartranslocation (see: WO 01/15511).

Other examples of moieties which may facilitate transfer into a targetcell are described in U.S. Pat. No. 6,204,054, which includestranscytosis vehicles and enhancers capable of transportingphysiologically-active agents across epithelia, endothelia andmesothelia containing the GP60 receptor. The GP60 receptor has beenimplicated in receptor-mediated transcytosis of albumin across cellbarriers. U.S. Pat. No. 6,204,054 exploits GP60 receptor-mediatedtrauscytosis for the transport of physiologically-active agents which donot naturally pass through epithelia, endothelia and mesothelia via theGP60 system. The pharmacophore can be coupled to albumin, albuminfragments, anti-GP60 polyclonal and monoclonal antibodies, anti-GP60polyclonal and monoclonal antibody fragments, and GP60 peptide fragmentsto facilitate transport into the cell.

Conjugation to a functional group may also improve other properties ofthe pharmacophore. Such functional groups may be termed drug carriersand can improve the solubility, stability, or biocompatibility of thedrug being carried. For example the solubility of the pharmacophore maybe improved by conjugating the pharmacophore to a peptide polymer. Byway of example U.S. Pat. Publication No. 2001041189 describes the use ofpolypeptides (containing glutamic acid and aspartic acid, or glutamicacid/alanine, or glutamic acid/asparagine, or glutamic acid/glutamine,or glutamic acid/glycine) conjugated to hydrophobic drugs such aspaclitaxel to act as carriers to improve the solubility of the drugsand/or their therapeutic efficacy in vivo. Similarly, U.S. Pat. No.5,087,616 describes the use of a biodegradable polymeric carrier (ahomopolymer of polyglutamic acid) to which one or more cytotoxicmolecules, such as daunomycin is conjugated Also by way of example, U.S.Pat. No. 4,960,790 describes paclitaxel covalently conjugated to anamino acid (glutamic acid) to improve drug solubility. Another exampleis described in U.S. Pat. No. 5,420,105, where polypeptide carrierscapable of binding one drug or multiple drugs can further be attached toa targeting or delivery protein, such as an antibody or ligand capableof binding to a desired target site in vivo.

Another example of a drug carrier is described in U.S. Pat. PublicationNo. 2001034333, where cyclodextrin polymers are used for carrying drugsand other active agents for therapeutic, medical or other uses. The2001034333 specification also discloses methods for preparingcompositions of cyclodextrin polymer carriers that are further coupledto delivery and targeting molecules to deliver drugs, like paclitaxeland doxorubicin, to their site of action.

By way of a further example, U.S. Pat. No. 6,127,349 describes the useof phospholipids to improve the solubility of the therapeuticagents(steroids, peptides, antibiotics and other biologically activeagents and pharmaceutical formulations) and to improve theirbio-availability. Similarly, fatty acids could be conjugated to thepharmacophore in order to stabilize the activity of the anti-angiogenicsubstances. By way of example U.S. Pat. No. 6,380,253 describes theconjugation of anti-angiogenic substances (proteins—angiostatin andendostatin etc.) to cis-unsaturated fatty acids or polyunsaturated fattyacids to potentiate and stabilize the activity of the anti-angiogenicsubstances.

Other suitable drug carriers include biologically compatible polymerssuch as polyethylene glycol (PEG) and related polymer derivatives.Drug-PEG conjugates have been described as improving the circulationtime (prolong serum half-life) before hydrolytic breakdown of theconjugate and subsequent release of the bound molecule thus increasingthe drugs efficacy. For example, U.S. Pat. No. 6,214,966 describes theuse of PEG and related polymer derivatives to conjugate to drugs such asproteins, enzymes and small molecules to improve the solubility and tofacilitate controlled release of the drug. Alternatively, EP 1082105 (WO99/59548) describes the use of biodegradable polyester polymers as adrug delivery system to facilitate controlled release of the conjugateddrug.

As another alternative the pharmacophore may be conjugated to anotherpharmaceutically active compound to enhance the therapeutic effect onthe target cell or tissue by delivering a second compound with a similaranti-mitotic effect or a different activity altogether. For example,U.S. Pat. No. 6,051,576 describes the use of co-drug formulations byconjugating two or more agents via a labile linkage to improve thepharmaceutical and pharmacological properties of pharmacologicallyactive compounds.

Compounds of this invention may be made from eleutherobin andeleuthoside compounds isolated from natural sources, such as isdescribed in WO 01/38339. Alternately, compounds of this invention maybe prepared by total synthesis by adapting the methods of Nicolaou, K.C., et al. [supra] and those disclosed in WO 01/38339 using conventionalstarting materials, or from intermediates prepared by reduction andglycosylation of sarcodictyin A (see: WO 96/14745). Intermediates usedin the preparation of compounds of this invention may includeisoeleutherobin A, desmethyleleutherobin or eleutherobin, withappropriate substitutions at R¹⁻³ done using conventional procedures.

At one time, the only known natural source of eleutherobin was a speciesof soft coral from Western Australia (see: Lindel, T. et al. [supra]. WO01/38339 disclosed an abundant new source of antimitotic terpenoids froma taxonomic order of coral and coral-like organisms much different fromthe order Alcyoniidae which comprises the soft coral described byLindel, T. et al. Using assays specifically adapted to detectantimitotic compounds, it was determined that organisms of the orderGorgonacea produce such antimitotic compounds. Such organisms includespecies of the genus Erythropodium; species of the genus Rumphella(family Gorgoniidae); Mopsea whiteleggei and Muricellisis Sp. a (familyIsididae); Subergorgia Sp. 1 cf Mollis and Subergorgia Mollis (geog.variant) (family Subergorgiidae); and, Junceella Sp. d. Verrucella Sp. band Ctenosella regia (family Ellisellidae).

A preferred natural source of intermediate compounds for use inpreparing compounds of this invention are the gorgonian corals, and inparticular, Erythropodium caribaeorun. Gorgonian corals are found in alltropical and sub-tropical regions, particularly the Caribbean. Thesecorals are found in abundance, and have been grown in aquariumenvironments and may be readily identified (for example, see: Bayer, F.M.; “The Shallow-Water Octocorallia of the West Indian Region” (1961)Martinus Nighoff; The Hague, at page 65 and 75–77 for Erythropodium). E.caribaeorum may be collected in abundance from southern Florida to theVirgin Islands.

Methods suitable for assaying antimitotic activity of compounds of thisinvention or compounds used as precursors, may be based on the use ofantibodies specific for mitotic cells, such as those described in theinternational patent application published Apr. 1, 1999 under WO99/15157 or the assay described in WO 01/38339. Such an assay willtypically employ cells which regularly divide in culture (e.g. cancercells). A known antimitotic compound such as nocodazole may be used as acontrol. In the assay, determination of the cells which proceed tomitosis is carried out using any of the known immunological methods byemploying antibodies which have specificity for mitotic cells.Monoclonal antibodies demonstrating such specificity are known andinclude MPM-2 which was raised against mitotic HeLa cells and recognizesphospho-epitopes that are highly conserved in mitotic proteins of alleukaryotic species. Other examples are the monoclonal antibodiesrecognizing phospho-epitopes in the paired helical filament proteins(PHF) found in brain tissue of patients suffering from Alzheiner'sdisease as described in: PCT International Application published Jul. 4,1996 under No. WO 96/20218; and, Vincent et al. (1996) “The Journal ofCell Biology”, 132:413–425. TG-3 antibody described in the latter tworeferences may be obtained from Albert Einstein College of Medicine ofYeshiva University, Bronx, N.Y. This antibody is highly specific formitotic cells and functions in ELISA.

Immunological methods useful for determination of mitotic cells in anassay include any method for determining antibody-antigen binding,including: immunocytochemistry (e.g. immunofluorescence), flowcytometry, immunoblotting, and ELISA, including those described inVincent, I. et al. [supra]. High throughput testing of samples may bereadily achieved by use of the ELISA or the ELICA assays described in WO01/38339.

Pharmaceutical preparations containing compounds of this invention maybe prepared as for similar preparations containing eleutherobin,paclitaxel, etc. In the case of compounds of this invention capable ofsalt formulation, pharmaceutically acceptable salts may be used toadvantage to permit administration of the compound in an aqueoussolvent. Modes of administration to an animal or human patient includeintravenous and intraperitoneal, to achieve a circulating concentrationof the drug as predicted from its activity using standard methodology.

A human or other animal patient suffering from proliferative diseases,and other similar conditions may be treated by administering to thepatient an effective amount of one or more of the compounds of thisinvention or a pharmaceutically acceptable derivative or salt thereof,in a pharmaceutically acceptable carrier or diluent. The activematerials can be administered by any appropriate route, for example,orally, parenterally, intravenously, intradermally, or subcutaneously.

The term pharmaceutically acceptable salts or derivatives refers tosalts or complexes that retain the antimitotic activity of the compoundand exhibit minimal undesired toxicological effects. Nonlmiting examplesof such salts are (a) acid addition salts formed with inorganic acids(for example, hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid, and the like), and salts formed withorganic acids such as acetic acid, oxalic acid, tartaric acid, succinicacid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,naphthalenedisulfonic acid, and polygalacturonic acid; (b) base additionsalts formed with polyvalent metal cations such as zinc, calcium,bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium,sodium, potassium, and the like, or with an organic cation formed fromN,N-benzylethylene-diamine, D-glucosamine, ammonium, tetraethylammonium,or ethylenediamine; or (c) combinations of (a) and (b); e.g., a zinctannate salt or the like.

A compound of this invention or salt thereof, may be included in apharmaceutically acceptable carrier or diluent, ideally in an amountsufficient to deliver to a patient a therapeutically effective amountwithout causing serious toxic effects in the patient treated. Theconcentration of active compound in the drug composition will depend onabsorption, distribution, inactivation, and excretion rates of the drugas well as other factors known to those of skill in the art. It is to benoted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgement ofthe person administering or supervising the administration of thecompositions.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application may include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftoxicity such as sodium chloride or dextrose.

Suitable pharmaceutically acceptable carriers for parenteralapplication, such as intravenous, subcutaneous, or intramuscularinjection, include sterile water, physiological saline, bacteriostaticsaline (saline containing 0.9 mg/ml benzyl alcohol) andphosphate-buffered saline. If administered intravenously, preferredcarriers are physiological saline or phosphate buffered saline (PBS).Methods for preparing transdermal patches are known to those skilled inthe art. For example, see Brown L., and Langer R., Transdermal Deliveryof Drugs (1988), Annual Review of Medicine, 39:221–229.

Compounds of this invention may be prepared with carriers that willprotect the compound against rapid elimination from the body, such asthrough controlled release formulations, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

Liposomal suspensions are also suitable carriers for compounds of thisinvention. The compounds may be conjugated to a lipid by known methodsfor incorporation into a liposomal envelope or the compounds may beencapsulated into the liposome. Liposomes may be prepared according tomethods known to those skilled in the art, such as is described in U.S.Pat. No. 4,522,811. For example, liposome formulations may be preparedby dissolving appropriate lipid(s) (such as stearoyl phosphatidylethanolamine stearoyl phosphatidyl choline, arachadoyl phosphatidycholine, and cholesterol) in an inorganic solvent that is thenevaporated, leaving behind a thin film of dried lipid on the surface ofthe container. An aqueous solution of the active compound or itsmonophosphate, and/or triphosphate derivatives are then introduced intothe container. The container is then swirled by hand to free the lipidaggregates, thereby forming the liposomal suspension.

Oral compositions may include an inert diluent or an edible carrier.They may be enclosed in gelatin capsules or compressed into tablets. Forthe purpose of oral therapeutic administration, the active compound canbe incorporated with excipients and used in the form of tablets,troches, or capsules. Pharmaceutically compatible binding agents, and/oradjuvant materials can be included as part of the composition. Methodsfor encapsulating compositions (such as in a coating of hard gelatin)for oral administration are well known in the art (Baker, Richard,Controlled Release of Biological Active Agents, John Wiley and Sons,1986).

Tablets, pills, capsules, troches and the like can contain any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, gum tragacanth or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,or corn starch; a lubricant such as magnesium stearate; a glidant suchas colloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavouring agent such as peppermint, methyl salicylate,or orange flavouring. When the dosage unit form is a capsule, it cancontain, in addition to material of the above type, a liquid carriersuch as a fatty oil. In addition, dosage unit forms can contain variousother materials which modify the physical form of the dosage unit, forexample, coatings of sugar, shellac, or other enteric agents.Alternatively, compounds of this invention could be administered as acomponent of an elixir, suspension, syrup, wafer, chewing gum or thelike. A syrup may contain, in addition to the active compounds, sucroseas a sweetening agent and certain preservatives, dyes and colourings andflavours.

EXAMPLES

Synthesis from Eleutherobin

Example 1

The first objective was to trap the C-4 ketone in eleutherobin as acyclic ketal in order to liberate the C-4/C-7 ether oxygen as a C-7alcohol and to change the oxygen atom's spatial relationship with theC-14 isopropyl group. In order to test the reactivity of the C-4hemiketal, desmethyleleutherobin 2 was treated with various neataliphatic alcohols (ROH: ethanol, n-propanol, n-butanol or isopropanol)and excess pyridinium p-toluenesulfonate (PPTS) at room temperature(rt), which gave quantitative conversion to the corresponding C-4 ketalanalogs (Table 1). Attempts to make the C-4 cyclic ketals of 2 withethylene glycol or 2,2-dimethyl-1,3-propanediol under a variety ofconditions using PPTS as a catalyst only gave the ketals 3 and 4 whichwere comparable in antimitotic activity (IC₅₀=20 nM and 80 nMrespectively) to eleutherobin (IC₅₀20 nM). The x-ray structure ofeleutherobin shows significant distortion of the C-1/C-2/C-3 (132{hacekover (s)}) and C-2/C-3/C-4 (127{hacek over (s)}) bond angles. This anglestrain may act like a clamp to keep the dihydrofuran ring from openingduring the transketalization reactions.

TABLE 1

1 R = Me

Example 2

Oxidation reactions involving the Δ11,12 olefin of eleutherobin 1 wereperformed (Table 2). Reaction of 1 with m-chloroperbenzoic acid (MCPBA)in CH₂Cl₂ at rt for 8 h gave a mixture of only two epoxides, 5 and 6.The 1H NMR data obtained for both 5 and 6 showed the absence of aresonance that could be assigned to H-12 and in both spectra the Me-17resonance had undergone a significant upfield shift. The observation ofa 1D NOESY correlation between the Me-17 resonance at δ and the H-2resonance at δ showed that the major epoxide was the β isomer 5.Treatment of 1 with SeO₂ (0.5 equiv.) in refluxing ethanol (EtOH) gave asingle product 7 in modest yield. The α and β epoxides 5 and 6 (whichare compounds of formula II of this invention) and the17-hydroxyeleutheside 7 (which is a compound of formula I of thisinvention) all had antimitotic potencies comparable to eleutherobin. TheIC₅₀ values on the above-described cell based mitotic assay were 300 nM,30 nM and 20 nM for compounds 5, 6, and 7 respectively. Compounds 5 and6 are particularly suited for use as intermediates given the stable butreactive nature of the epoxide functionality. Compound 7 is particularlysuitable as an intermediate for the same reasons and is less hydrophobicthan eleutherobin, thereby allowing for the formulation of a moresoluble pharmaceutical preparation of the compound, as compared toeleutherobin.

TABLE 2

Example 3

Reduction of the olefins in 1 was examined. A solution of eleutherobinin ethyl acetate (EtOAc) containing catalytic palladium (Pd) on BaSO4was stirred at rt for 1 h under 1 atm of H₂ resulting in the formationof hexahydroeleutherobin 8 (Table 2). The 1H NMR data obtained for 8,which contained only a single olefinic proton resonance at δ 5.78 (d,J=9.3 Hz) assigned to H-2, clearly indicated that the Δ5,6, Δ11,12, andΔ2′,3′ double bonds had been reduced. A 1D NOESY enhancement observedbetween the Me-17 resonance at δ 0.76 (d, J=7.0 Hz) and the H-2resonance at δ 5.78 demonstrated that hydrogen had added to the moreaccessible β face of the Δ11,12 olefin. Hexahydroeleutherobin 8(IC₅₀>10⁴ nM was found to be more than five thousand fold less activethan eleutherobin, indicating that the presence of one or more of thereduced double bonds affects tubulin binding. Previous evaluation of asynthetic sarcodictyin library had suggested that reduction of the Δ5,6olefin had minimal effect on the potency of tubulin polymerization.(Nicolaou, K. C. et al. (1998) J. Am. Chem. Soc. 120:10814–26).Therefore, 5,6, 11,12-tetrahydroeleutherobin (9) and2′,3′-dihydroeleutherobin (10) were selected as targets to further probethe biological effects of olefin reduction.

Example 4

The synthesis of 9 started from eleutherobin, through first forming the3″,4″-acetonide 11, which was subsequently deacetylated to the 2″alcohol and converted directly to the 2″ TBS ether 12. Hydrogenation of12, using catalytic Pd on BaSO4, gave the 5,6,11β,12,2′,3′-hexahydroderivative vide supra (Table 3). Hydrolysis of the crude hydrogenationproduct cleanly removed the C-8 ester side chain, which was replacedwith a urocanic ester residue 4c to afford 14. Deprotection of thetribromosalicylanilide (TBS) protecting group, followed by acetylationof the formed 2″ alcohol provided 15, which was subsequently deprotectedunder mildly acidic conditions to give 5,6,11β,12-tetrahydroeleutherobin (9). Similarly, 2′3′-dihdro-eleutherobin (10) was preparedfrom the 2″ TBS ether 12, by hydrolysis of the urocanic ester residue(NaOH, MeOH) to provide a secondary alcohol at C-8, which was directlycoupled with 2,3dihydro-urocanic acid (17) using1,3-dichlorohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP)in warm (50° C.) dimethyl formanide (DMF) to afford 15. Adeprotection/acetylation sequence similar to that employed in thesynthesis of the tetrahydro derivative 9, provided 10 in good yield.Compound 9 exhibited antimitotic activity (IC₅ 200 nM) comparable toeleutherobin while compound 10 demonstrated drastically reducedantimitotic activity (IC₅₀ 20000 nM). This demonstrates that reductionof the Δ^(2′,3′) double bond is responsible for loss of antimitoticactivity.

Example 4

The synthesis of 9 started from eleutherobin, through first forming the3″,4″-acetonide 11, which was subsequently deacetylated to the 2″alcohol and converted directly to the 2″ TBS ether 12. Hydrogenation of12, using catalytic Pd on BaSO4, gave the 5,6,11β,12,2′,3′-hexahydroderivative wide supra (Table 3). Hydrolysis of the crude hydrogenationproduct cleanly removed the C-8 ester side chain, which was replacedwith a urocanic ester residue 4c to afford 14. Deprotection of thetribromosalicylanilide (TBS) protecting group, followed by acetylationof the formed 2″ alcohol provided 15, which was subsequently deprotectedunder mildly acidic conditions to give 5,6,11β,12-tetrahydroeleutherobin (9). Similarly, 2′3′-dihdro-eleutherobin (10) was preparedfrom the 2″ TBS ether 12, by hydrolysis of the urocanic ester residue(NaOH, MeOH) to provide a secondary alcohol at C-8, which was directlycoupled with 2,3-dihydro-urocanic acid (17) using1,3dichlorohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) inwarm (50° C.) dimethyl formanide (DMF) to afford 15. Adeprotection/acetylation sequence similar to that employed in thesynthesis of the tetrahydro derivative 9, provided 10 in good yield.Compound 9 exhibited antimitotic activity (IC₅₀ 200 nM) comparable toeleutherobin while compound 10 demonstrated drastically reducedantimitotic activity (IC₅₀ 20000 nM). This demonstrates that reductionof the Δ^(2′,3′) double bond is responsible for loss of antimitoticactivity.

TABLE 3

Selective Modifications at C-17

Labelling of substituents (e.g. R³–R⁶) in the structural formulae setout in Tables 4–8 below is not the same as the labelling of thecorresponding substituents (e.g. R₁–R₄) in the definition of compoundsof this invention as set out above. Labelling used in the followingTables is for convenience in following the synthesis schemes describedin Examples 5–9. In any case, substituents of compounds of thisinvention may be recognized by their position in a structural formulaand by reference to the description above.

Example 5

As demonstrated in Example 2, the C-17 methyl group of eleutherobin isreadily oxidized with SeO₂ in refluxing ethanol to provide an allylicalcohol at C-17. This reaction is again shown in Table 4 with compound(X) representing eleutherobin or an analog thereof to produce compound(XV). Using compound (XV) as an intermediate, a number of reactions maybe undertaken to further modify the functionality at C-17 (Table 4).These reactions are preferably undertaken when the C-3″ and C-4″ OH'sare protected as an acetonide or in some cases as ester. Examples ofprocedures for protecting and deprotecting the sugar OH's are givenbelow. The allylic alcohol XV can be converted to esters XXIV byreaction with anhydrides. As outlined in Table 4, this alcohol may alsobe converted to a halide (PPh₃, imid, X₂), to provide an allylic halideXVI. This allylic halide may be displaced by a number of nucleophilesunder standard conditions to provide compounds of general structureXVII. In addition, the allylic alcohol may be oxidized to an aldehyde(TPAP, NMO, CH₂Cl₂) such as XVIII, and further oxidized (t-butanol,2-methyl-2-butene, NaH₂PO₄, NaClO₂, H₂O) to the corresponding carboxylicacid (XIX). Examples of simple modifications of both the aldehyde XVIIIand the acid XIX are set out in Table 4. As the aldehyde functionalityin XVIII represents the most electrophilic centre in the molecule, itmay be coupled with a variety of Wittig type nucleophiles under standardconditions to provide alkenes of general structure XX. Alternatively,the aldehyde functionality may be coupled with a number of standardnucleophiles to provide a secondary alcohol of general structure XXI.The carboxylic acid functionality in XIX may also be selectively reactedwith various alcohols (to provide esters XXII) or amines (to provideamides XXIII). Modifications of the C-17 methyl moiety (and thesubsequent modifications to the aldehyde XVIII and the acid XIX) thatare described above may also be applied to compounds in whichsubstitutions and modifications at C-4, and C-2″, C-3″ and C-4″ of thesugar moiety have also been undertaken.

TABLE 4 Selective Modifications at C-17

Example 6

Some transformations described in Example 5 rely primarily on theability to efficaciously transform the substituents on the sugarappendage at C-15 of eleutherobin. Concurrent protection of the C-3″ andC-4″ hydroxyl groups of eleutherobin (I) to provide eleutherobinacetonide (II) are possible as is shown in Table 5. Eleutherobinacetonide (II) may be an intermediate for use in further modifications.A sequence of reactions which allows access to a uniquely substitutedsugar is described in Table 5. The acetate appendage at C-1″ ineleutherobin acetonide may be selectively hydrolized to provide III,which can be coupled with a variety of protecting agents (such astert-butyldimethylsilylchloride) to provide a structure such as IV. Withthe sugar suitably protected, the core (C-1 to C-20) of eleutherobin maybe further functionalized or modified. In particular, substitutions ofvarious polar, nonpolar and bulky alkyl groups for the methyl ketal atC-4 (R₃), and functionalization of the methyl group (R₁) and replacementof the N-methyl urocanic ester residue (R₂) at C-8 with a variety ofα,β-unsaturated esters provides expedient access to new series ofeleuthesides, As outlined in Table 5, with various substitution patternsat the C-4 ketal, the C-8 hydroxyl and the methyl at C-17, theprotection group at C-2″ may be removed under standard conditions toprovide a free hydroxyl which can then be subsequently acetylated toafford compounds of general formula VI. The remaining acetonideprotecting group is cleanly removed under mildly acidic conditions toprovide a compound in which the sugar moiety is identical to that foundin eleutherobin with the core (C-1 to C-20) capable of being modified asdescribed below.

TABLE 5 Protection/deprotection of Eleutherobin (I)

Example 7

As outlined in Example 6, the sugar appendage may be selectivelyprotected and further functionalized. An intermediate such as IV may bedeprotected through an alternative sequence (Table 6) whereby access toa sugar moiety selectively substituted at C-2″, C-3″ and C-4″ is gained.Initial deprotection of the C-3″ and C-4″ acetonide provides access to acompound of general structure VI. Taking advantage of the inherentdifferences in reactivity between an axial (C-4″) hydroxyl and anequatorial (C-3″) hydroxyl, the substituents at both C-3″ and C-4″ maybe selectively introduced to provide a compound of general structureVIII. The protecting group at C-2″ can subsequently be removed andreplaced by a variety of substituents to provide X (Table 6) in whichthe sugar moiety is now uniquely functionalized at C-2″, C-3″ and C-4″.These modifications of the sugar moiety may also be applied to compoundsin which substitutions and modifications at C-4, C-8 and C-17, such asare described in Example 8, are undertaken.

TABLE 6 Selective Substitution on Sugar

Example 8

The methyl ketal functionality present in eleutherobin at C-4 may beconverted to a variety of bulky, polar or nonpolar ketals through atransketalization reaction catalyzed by PPTS. As depicted in Table 7,this transformation lends access to a series of new eleuthesides and canbe undertaken in situations where C-17, C-8 and the sugar moiety arevariously substituted.

TABLE 7 Selective Transketalization

Example 9

Table 8 sets out various examples of compounds of formula I of thisinvention which are made using standard techniques from an alcohol orcarboxylic acid moiety at C-11. Included are examples where moiety X ofcompounds of formula I includes a moiety (Q) intended to enhance in vivodelivery (e.g. a lipid such as is found in a lipid-based deliveryvehicle such as a liposome) or a biologically active moiety (such as anantibody or hormone intended to enhance targeting of the compound to acell type, cell receptor, etc.). Moiety Q may include a linker used tojoin a moiety such as a lipid, antibody, hormone, etc. to C-11. Suchlinkers may be designed to be cleaved in certain environments or underparticular conditions to enhance release of the antimitotic moiety fromQ at or in a target cell.

Compounds shown in Table 8 as coming from compounds 5 or 6 of Table 2are examples of compounds which may be readily made from compounds offormula II of this invention.

TABLE 8

All publications, patents and patent applications referred to herein arehereby incorporated by reference. While this invention has beendescribed according to particular embodiments and by reference tocertain examples, it will be apparent to those of skill in the art thatvariations and modifications of the invention as described herein.

1. A compound or pharmaceutically acceptable salt thereof, wherein thecompound is of formula I or II:

wherein, Me is methyl; R¹, R² and R³ are independently selected from thegroup consisting of: H and C₁–C₆ acyl; R⁴ is selected from the groupconsisting of: H,Me and a substituted or unsubstituted straight-chain,branched, or cyclic C₁–C₁₀ alkyl; and, X is selected from the groupconsisting of: —CH₂OR, —COR, —COY, —C(OR)R, —CRCR₂, and, —CH₂W; R isselected from the group consisting of: H; a linear, branched, or cyclic,saturated or unsaturated alkyl group containing one to ten carbonsoptionally substituted with Z; an aromatic group optionally substitutedwith Z; and, arylalkyl (ArR*) in which an alkyl group (R*) is a linear,branched, or cyclic, and saturated or unsaturated containing one to tencarbons a optionally substituted with Z and an aryl group (Ar)optionally substituted with Z; Z is selected from the group consistingof: —OH, —OR, —O₂CR, —SH, —SR, —SOCR, —NH₂, —NHR, —NHR₂, —NHCOR, —NRCOR,—I, —Br, —Cl, —F, —CN, —CO₂H, —CO₂R, —CHO, —COR, —CONH₂, —CONHR, —CONR₂,—COSH, —COSR, —NO₂, —SO₃H, —SOR, and —SO₂R; Y is selected from the groupconsisting of: —OH, —NH₂, —NHR, —NR₂, —SH, —SR and —OR; W is selectedfrom the group consisting of: R, F, Cl, Br, I, 13 OSO₃R, —SO₃R, —OPO₃R₂,—PO₃R₂, —SR, —SOR, —SO₂R, —NR₂, —NOR, and —NR₃ ⁺; with each R being thesame or different, and wherein for R* and for R when comprising analkyl, —CH₂— may be replaced by —O₂S or NR and CH may be replaced by N;and wherein W=R, R cannot be methyl or ethyl.
 2. A compound of claim 1,wherein R¹ and R² are H and R³ is acyl.
 3. A compound of claim 1,wherein one of R¹ and R² is H, and the other of R¹ and R² is acyl and,R³ is H.
 4. A compound of claim 1, wherein one or more of R¹–R⁴ isacetyl.
 5. A compound of claim 2 or 3, wherein acyl is acetyl.
 6. Acompound of claim 1, wherein one or more of R¹–R⁴ is a C₂–C₅straight-chain or branched alkyl.
 7. A compound of claim 1, wherein R⁴is selected from the group consisting of: H, ethyl, propyl, butyl andpentyl.
 8. A compound of claim 1, wherein R¹ and R³ are H; R² is acyl;and, R⁴ is methyl.
 9. A compound of claim 8, wherein R² is acetyl.
 10. Acompound of claim 1, wherein R is: H; or, a linear, branched or cyclic,saturated or unsaturated alkyl group; and, Y is selected from the groupconsisting of: OH, NH₂, NHR, NHR₂ and OR.
 11. A compound of claim 1,wherein R is H or a C₁–C₆ branched or straight chain alkyl.
 12. Acompound of claim 1, wherein X is selected from the group consisting of:—COH; —COOH; —CNH₂; —CNHR; —CNR₂; and, —COR.
 13. A compound of claim 12,wherein R is a C₁–C₆ branched or straight chain alkyl.
 14. A compound ofclaim 1, wherein X is —CH₂OH.
 15. A compound of claim 1, wherein thecompound is of formula II.
 16. A compound of claim 15, wherein theepoxide at C-11 is in a β configuration.
 17. A pharmaceuticalpreparation comprising a compound or pharmaceutically acceptable salt ofclaim 1 and a pharmaceutically acceptable carrier or diluent.